Hh pathway mutations sensitize embryos to ethanol‐ or PBO‐induced craniofacial defects. (A–D) Flat‐mount preparations of 5 dpf neurocrania showing the range of phenotypes observed. Phenotypes ranged from apparently normal to severe, with increasing severity determined by more extensive malformations of the bilateral trabeculae. All images were captured at 10X magnification. (A′–D′) 20X magnification of the right trabeculae of A–D shows cell arrangement defects in affected embryos, specifically the stacking defect in mild embryos (B′) compared to normal stacking (A′). (E–F) Wild‐type or embryos with a single‐allele mutation in shha (tq252) were exposed to 0% 0.75% or 1% ethanol (E) or 0, 6.25, or 12.5 μM PBO (F). Percent malformations (mild, moderate, and severe) are shown. Incidence of malformations was compared between genotypes for each treatment group using Fisher’s exact test with Bonferroni correction for multiple comparisons. N ≥ 15 embryos per genotype per treatment.

Ethanol and PBO synergistically interact to cause craniofacial defects. (A) Wild‐type embryos were exposed to 0, 6.25, 12.5, or 25 μM PBO with or without a 1% dose of ethanol. Percent malformations (mild, moderate, and severe) are shown. Incidence of malformations was compared between treatment groups using Fisher’s exact test with Bonferroni correction for multiple comparisons. ***p < 0.001, **p < 0.01. (B–F) Whole‐mount images of alcian‐ and alizarin‐stained embryos are shown for each treatment group. (G) 5‐day‐old alcian‐ and alizarin‐stained embryo marks the measurement for inter‐trabecular width (white dashed outline). (H) Quantification of inter‐trabecular widths. Mean width ± SEM is shown. Measurements were normalized to control and compared using one‐way ANOVA with Tukey’s multiple comparisons test. Different letters indicate statistically significant differences between the groups. See supplemental data for the specific multiple comparisons p‐values. N ≥ 28 embryos per treatment.

Mutations in shha sensitize embryos to multifactorial interactions between ethanol and PBO. (A) Wild‐type (+/+) or heterozygous (+/‐) embryos for shha (tq252) were exposed to subthreshold doses of PBO (3.125 μM), ethanol (0.5%), or the combination of both chemicals (3.125 μM PBO + 0.5% ethanol). Percent malformations (mild, moderate, and severe) are shown. Incidence of malformations was compared between genotypes for each treatment group using Fisher’s exact test with Bonferroni correction for multiple comparisons. (B) Quantification of inter‐trabecular widths. Mean width ± SEM is shown. Measurements were normalized to control and compared using one‐way ANOVA with Tukey’s multiple comparisons test. Different letters indicate statistically significant differences between the groups. See supplemental data for the specific multiple comparisons p‐values. N ≥ 14 embryos per genotype per treatment.